New vitamin b factor



May 2, 1961 K. BERNHAUER ET A1. 2,982,766

NEW VITAMIN B'z FACTOR Filed July 22, 1957 :s sheets-sheet 1 SV/57 I May 2, 1961 K. BERNHAUER ET AL NEW VITAMIN BIZ FACTOR FIC 7025 WEE-72 INVENTORS WMM' @elif/7' May 2, 1961 K. BERNHAUER ET AL 2,982,766

NEW VITAMIN Bl2 FACTOR Filed July 22, 1957 3 Sheets-Sheet 3 SHEET .3

INVENTORS ASMI/rnv( kww dw( Y NEW B12 FACTOR Konrad fBernhauer, yAschalenburg (Main), and Hanswerner Dellweg, Stockstadt (Main), Germany, assignors, by mesne assignments, to Merck & Co., inc., Rahway, N.J.` Y

Filed July zz, 1951, ser. No. 673,433 claims priority, application Germany July 21, 1956 1 claim; (c1. 26o-234) The present invention relates to the production and recovery of a new vitamin B12 factor, and more particu;

yCN* 1 Aetiocobal'amine (without nucleotide) Aetiocobalamine-phosphoric acid (without nucleoside) v III Oe-P-OH d-z'ilbose n Aetocobalaxninephosphoric acid-ribose (withoutbase) United States Patent ICC d-ri osebase Complete Bm factor (without nucleotide) The complete structural formula of Product III, with which the present invention is concerned, is as follows:

CONH:

oN on2 HzNoooH,

if y

, y HOCH2- l Of the possible incomplete B12 factors the only one known prior to the present invention is vitamin B12 factor B (aetiocobalamineFormula I). As may be seen from the vstructural formulas, this compound ydiffers from the complete factor (Formula IV) onlyin the lack of a nucleotide. The other two theoretically possible factors were unknown prior to the present invention.

It is therefore a' primary object of the present invention to provide for the production lof a new incomplete vitamin B12 factor.

'It is another objective of the present invention to provid-e a new method for the production of the new in-A complete vitamin B12 factor of the present invention.

It is more particularly the basic object of the presentv invention to provide for the production of an incomplete vitamin B12 factor corresponding to the Formula III shown above.

Other objects and advantages of the present invention will be apparent from a further reading of the specication and of the appended claim.

Y. With the above objects in view, the present invention structural formula corresponding to Formula III above.

The novel features which are considered as characteristic for the invention are set forth in particular in the appended claim. The invention itself, however, with respect to the method thereof will be best understood from the following description of the method when read in conjunction with the accompanying drawings in which a iiow sheet illustrating the method of the present invention is set forth.

Sewage sludge, which asis known, serves as the source material for the production of vitamin B12 and other w'tamin B12 factors (German `Patent No. 922,126) can also serve as the source of the compound of the present invention.`

The method of the present invention comprises a special combination of various steps each of which per se may be known with the final purpose of producing the new prodnot of the present invention which is herein designated as aetiocobalamine-phospho-ribose." The attached iiow sheet indicates the individual method steps of the method of the present invention, the following description more fully describing the steps.

1 In accordance with'the method of the invention digested sewage sludge is first 'treated with about 0.1% of sulfur dioxide, the pH is adjusted to about 6.5 and the mass is heated to temperature of :80 C. About 0.5% of ferric chloride is added and the mass is centrifuged. The centrifugate is acidiiied to a pH of 3.0, the pH is then adjusted to f6.5 to vcause flocculation of colloidal substances and this mass is again centrifuged. The clear filtrate is mixed with 0.5% of activated carbon and the material is again centrifuged. The carbon adsorbate (solid material) is then eluted several times with a 1:1 mixture of water and isopropanol at about 70 C. The material is again centrifuged-and the eluate concentrated under vacuum. y

The eluate concentrate is `then purified by extraction, for example with phenol-'i-o-dichlorobenzol. The purified extraction concentrate is precipitated on kieselguhr by means of a phenol and this crude kieselguhr product is subjected to a iirst column chromatography on cellulose with secondary butanol and water, containing potassium chlorate and hydrogen cyanide. r[his results in separation of the new factor of the present invention between the vitamin B12 and vitamin B12 factor I-II zones. This new factor is then precipitated on kieselguhr by means of a phenol and subjected to a second column chromato graphy on cellulose with water-saturated n-butanol plus hydrogen cyanide to obtain the purified .new factor of the present invention.

This purified new factor is subjected to electrophoresis at pH 6.5 in cellulose. The fraction which migrates to the anode is the new vitamin B12 factor of the present invention. This fraction is eluted with phosphate buffer and subjectedto a rst chromatography on aluminum oxide. The eluate is evaporated under vacuum and the residue precipitated with zinc chloride at a pH of 8.5.

After filtration and washing the ltrate is treated with ammonium sulfate and extracted with benzyl alcohol,

the new factor is tranfserred intoY water and precipitated on kieselguhr by means of phenols. This material is then subjected .to a second chromatorgraphy on aluminum oxide, the eluate is evaporated under vacuum, the residue taken up in methanol, precipitated with acetone, subjected to suction filtration and then dried to obtain lthe new factor of thepresen't invention. v The new vitamin B12 factor of the present invention produced as described above has the following properties: The compound is a violet colored rather hygroscopic powder.

The absorption spectrum at pH 6.5 in the 'presence of cyanide ions is identical with the `spectrum of aetiocon under vacuum to a volume of 100 liters.

balamine (Formula I). The molar extinction at 367 mn Em1 =30.'59' 103.

'Iihe following are the R1-values in the indicated developer systems:

0.31 in G (sec. butanol saturated with water+0.01%

HCN saturated with KClOr);

0.33 in I (sec. butanol-100, water-36, 25% NH3-14 {-0.005% HCN).

Paper electrophoresis: pH 23h-neutral, orange; pI- I 6.5-monobasic acid, voilet. A Y A Y; n

The distribution coefficient equals 1 in the system of n-butanol `aqueous ammonium sulfate solutionv with 6.5% ammonium sulfate (dicyano form). 'A

The analysis and structure 'determinationsfof-'th'e 4new factor of the present invention yu'eld the following:

By decomposition with ,70% perchloric acid factor B is obtained, which can be identified by chromatographic and electrophoretic methods.

Decomposition kby means of cerium hydroxide results in factor B (identiiied as above) and dribose, which can be identified by paper chromatography.

The cobalt content was determined by the method o I. M. Chilton (Anal. Chem. 25, 1247, 1953). The phosphate content was determined according to the method 'of C. H. Fiske, Y. Subbarow (Methods of Biochem. Anal., Interscience Publishers Inc., New York 1954, vol. I, "S. 99). The ribose content was determined by means of the orcin reaction in accordance 'with the lmethod of A. H. Brown (Methods of Biochem. Anal., Interscience Publishers Inc., New York 1954, vol. I, page 298)-l Prom these determinations it is concluded that the new factor of the present invention consists ofl mol offactor B plus 1 mol of phosphoric acid plus 1 mol of ribose combined and that the compound has a molecular weight of 1255.23. The compound may therefore be designated as factor B-phospho-ribose (or aeticobalamine-phosphoribose) The new compound of the present invention has an activity of 13.5 'y/opt. E. against E. coli. The new factor can he decomposed into factor B, which in usual manner can be converted by biosynthesis into vitaminBm itself or other'vitamin B12 factors'vwhereby ity is possible to make additional use of the new factor of the present invention. i

The following example will further illustrate the method of producing the new compound of the present inven-- tion. The scope of the invention is of course not meant to be limited to the speciic details of the example.

Example 8 cubic meters of digested sludge,robtained by the methane fermentation of sewage sludge, afterthe addition of 0.1% SO2 and the adjustment of the pH value to 6.5 is heated at C. After the addition of 0.5% ferric chloride the solid material is centrifuged off, the

. centrifugate is :clarified by acidification to .pH 1310 .fand

flocculated turbidity-forming substances are eliminated after again adjusting the pH value to 6.5.

The obtained aqueous solution having a volume of 7.5 cubic meters is treated for adsorption of the vitamins of the B12 group with activated carbon. After obtaining the adsorbate by centrifugation the adsorbate is eluted at a temperature of about 70 C. with a mixture of equal parts of isopropyl alcohol and water. Y

The obtained eluate (about 900 liters) is concentrated The further purification proceeds by countercurrent extraction with o-dichlorobenzol and' p-chlorophenol in the same manner as described in German Patent No. 961,023, namely in a four-stage process in which the concentrated eluate is extracted in the rst stage with the solution of a halogenized phenol in a liquid hydrocarbon, for 'with a'halogenizcd hydrocarbon, or with other solvents such as carbon disulfide, or a mixture of such solvents; the thus obtained organic phase is then washed in the second stage with buffered solutions within the pH range 2-8.5, and with distilled water W-hereafter the vitamins are transferred in the third stage from the organic to the aqueous phase by addition of an oxygen-containing polar substance such as acetone and washed in the fourth stage With a suitable organic solvent which is immiscible with Water. 'Ihe obtaining of the kieselguhr product" is in accordance with the publication of W. Friedrich and K. Bernhauer (Z. Naturforschg. 9b, 755, 1954), whereby the oily vitamin B12-phenol complex is accepted by kieselguhr, forming a homogeneously red colored product which precipitates easily, whereby the supernatant liquid becomes clear. The kieselguhr product can be easily sucked off and washed with phenol-saturated water.

The column chromatography of the kieselguhr product no cellulose with a mixture of 77% sec. butanol, 23% water, 0.05% hydrocyanic acid saturated with potassium perchlorate (in accordance with German Patent No. 944,216) results in a violet band of the new factor between the zones of vitamin B12 and vitamin B12 factor III. The corresponding eluate from the column is again precipitated on kieselghur and subjected to chromatography on a cellulose slurry column utilizing watersaturated n-butanol as the developer. This results in a further separation from vitamin B12 and vitamin B12 factor III.

The fraction of the new factor which still contains some vitamin B12 and vitamin B12 factor HI as impurities (7350 opt. E.) is subjected to an electrophoretic separation. This is carried out in a cellulose column having a diameter of 60 mm. and a height of 400 mm. at 750 volts and 150 to 200 milliamperes with 0.0166 molar of phosphate buler at a pH of 6.5 during a time period of 12.5 hours. Under these conditions the new factor quickly migrates to the anode while vitamin B12 and vitamin B12 factor III because of electroosmosis migrate weakly to the cathode. The column is subsequently eluted with phosphate buffer.

After the addition of 6 times the amount of acetone to this solution the new factor (5400 opt. E.) is absorbed` on an aluminum column from which it is again eluted with a mixture of 70 parts acetone and 30 parts water. After concentration of the eluate under vacuum the residue is taken up in 200 cc. of water, mixed with 6 g. of zinc chloride and the pH value of the solution is adjusted to 8.5 by means of sodium hydroxide. The resulting precipitate is ltered oi by suction over kieselguhr and thoroughly washed with water. After the addition of 60 g. of ammonium sulfate to the red filtrate (300 cc.) the new factor is extracted with a total of 150 cc. of benzyl alcohol, the alcoholic extract mixed with chloroform and again extracted with water.

After an additional chromatography on aluminum oxide with acetone-water (80:20) as eluting agent, the obtained solution is evaporated to dryness under vacuum, the residue taken up with cc. of methanol and this mixed with 150 cc. of acetone. This results in occulation of the new factor of the present invention which after standing for 3 hours at a temperature of 6 -2 C. is completely flocculated. The obtained violet product is filtered off under suction with complete exclusion of air moisture, and it is then dried under vacuum over magnesium perchlorate.

The yield amounts to 234 mg., the purity of which is 65.14% calculated on the cobalt content and related to a molecular weight of 1255 .123.

Without further analysis, the foregoing will so fully reveal the gist of the present invention that others can by applying current knowledge readily adapt it for Various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specic aspects of this invention and, therefore, such adaptations should and are intended to be comprehended within the meaning and range of equivalence of the following claim.

What is claimed as new and desired to be secured by Letters Patent is:

As a new composition of matter, aeticobalamine-phosphoric acid-ribose having the following structural formula:

coun,

cN Bz bn, en, on, omooNH mucosal l B 4omorncoNrn C N*- C s NGK@ 'R N OE mNooon D OBs on, om ,om `omomooNm /Cz ON co N (g: 0e ont -o-P on 1!I 0/ \O n on (H H \0/ no n: n

References Cited in the le of this patent UNITED STATES PATENTS Miner et al. July 211, 1953 Lightfoot et al. Apr. 2, 1957 OTHER REFERENCES 

